As a medical diagnosis, pathological diagnosis is performed. A pathologist diagnoses a disease using a tissue section collected from human body and informs a clinician of whether or not a therapy and/or surgery is/are necessary. Based on the patient conditions and the pathological diagnosis, a physician determines pharmacotherapeutic strategies and a surgeon determines whether or not a surgery should be performed.
In pathological diagnosis, it is a common practice to prepare a tissue specimen by slicing a tissue sample obtained by evisceration or needle biopsy into a thickness of about several micrometers and then observe the tissue specimen at a magnification under a light microscope so as to obtain various findings. In many cases, a specimen is prepared by fixing a collected tissue through dehydration and paraffin blocking, slicing the thus fixed tissue into a thickness of several micrometers, and then removing the paraffin.
In pathological diagnosis, immunological observation in which molecular target staining called immunostaining is performed for verifying the expression of molecular information of a specimen and functional abnormalities such as abnormal expression of a gene or a protein are diagnosed is performed. For immunostaining, for example, a dye staining method using an enzyme (e.g., DAB staining) is employed. In DAB staining, an antibody modified with peroxidase, which is capable of allowing diaminobenzidine (DAB) to show a color, is used to stain an antigen to be observed with the color and the amount of the antigen is determined by observing the stained antigen. Alternatively, fluorescent labeling may be employed in some cases. In fluorescent labeling, the amount of the subject antigen is determined by staining the antigen with an antibody modified with a fluorescent dye and observing the stained antigen.
Further, since a specimen hardly absorbs or scatters any light and is thus nearly colorless and transparent, it may be subjected to staining with a die for morphological observation prior to being observed. There have been proposed a variety of staining techniques. In particular, for tissue specimens, hematoxylin-eosin staining (HE staining) using two dyes, hematoxylin and eosin, is typically used as staining for observing the morphology of the subject specimen (Non-patent Document 1, Patent Documents 1 and 2). Hematoxylin stains cell nuclei, calcareous parts, cartilaginous tissues, bacteria and mucus in livid to light blue, while eosin stains cytoplasm, interstitial tissues, various fibers, erythrocytes and keratinocyte in red to dark red. A pathologist makes a diagnosis based on the morphological and staining information, such as changes in the size and shape of cell nuclei and changes in the pattern as a tissue, in a micrograph of the stained tissue specimen. Examples of other staining for morphological observation include Papanicolaou staining (Pap staining) used for cytological diagnosis. By subjecting a tissue section to both morphological staining and immunostaining, morphological observation and immunological observation of the specimen can be performed simultaneously.
At present, DAB staining is often used in immunological observation (Patent Document 3). However, in staining with an enzyme label such as DAB staining, since the staining concentration is largely variable depending on the environmental conditions such as temperature and time, there is a problem that estimation of the actual amount of an antibody or the like from the staining concentration is difficult. Therefore, for immunological observation in pathological diagnosis, fluorescent labeling using a fluorescent label is also performed as an alternative to staining with an enzyme label. This method characteristically has superior quantitative capability than DAB staining (Non-patent Document 1).
Observation using a fluorescent label is performed under a confocal laser scanning microscope or epifluorescence microscope. Such microscopes utilize a high-intensity excitation light. For example, as opposed to an intensity of 1,000 W/m2, which is normal sunlight exposure test condition described in the solar cell standard JIS C 8914, the intensity of the irradiation light used by a typical epifluorescence microscope is 100 times stronger.
When the fluorescent label is damaged by excitation light and no longer emits light, a reduction in the signal occurs. Therefore, in the observation using a fluorescent label, the light resistance of the fluorescent label is important.
It is known that fluorescent dyes, inorganic nanoparticles (which may also be referred to as “semiconductor nanoparticle”, “quantum dot” or the like) and aggregates thereof are utilized as fluorescent labels (Non-patent Documents 2 and 3, Patent Document 4). It has been reported that, as compared to fluorescent dyes and inorganic nanoparticles, aggregates thereof have an improved light resistance (Patent Document 5). Therefore, from the standpoint of the light resistance, such aggregates are more preferred; however, the aggregates alone do not have sufficient light resistance required for fluorescence microscopy. Here, an aggregate has a higher brightness than that of one dye molecule; therefore, from the standpoint of the signal, an aggregate is more preferred.
In preparation of a specimen, aqueous mounting media and oil-based mounting media are known as mounting media for mounting a stained pathological section. Aqueous mounting media have a problem in that, since their refractive indices are largely different from that of a specimen, it is difficult to make a specimen transparent and to produce a permanent preparation thereof. Meanwhile, oil-based mounting media are characterized in that their refractive indices are not largely different from that of a specimen and can thus make the specimen transparent; and that they are typically used for preparing a permanent preparation that shows good color tone and color development in morphological staining. Accordingly, oil-based mounting media are more preferably used in the preparation of a specimen.
Therefore, also as a mounting medium for an immunostained specimen, a permanent preparation can be produced with an oil-based mounting medium and, when a specimen is subjected to double staining of immunostaining and morphological staining, it is believed that the use of an oil-based mounting medium showing good color tone and color development in morphological staining is more preferred.
However, when a fluorescently-stained specimen is mounted using an oil-based mounting medium, there is a problem that the fluorescent dye elutes into the mounting medium and this impairs the staining properties. Therefore, there is a circumstance that, while an oil-based mounting medium can be used when the specimen is stained without using any fluorescent label, an aqueous mounting medium having the above-described problems must be used when the specimen is stained with a fluorescent label.
In addition, for observation of a fluorescent label, a confocal laser microscope or a fluorescence microscope is used. In fluorescence observation under these microscopes, a stained section is irradiated with a high-intensity excitation light. This excitation light gradually deteriorates a fluorescent label containing a fluorescent dye or the like and this has a great effect in the fluorescence observation and assessment of immunostaining results. Ideally, there is no deterioration of such fluorescent label. Otherwise, it is necessary to improve the light resistance of the fluorescent label.
In order to improve the light resistance of a fluorescent label, it is thought to admix a discoloration inhibitor with a mounting medium. In aqueous mounting media, for example, incases where 4′,6-diamidino-2-phenylindole (DAPI) is used as a fluorescent dye, it is attempted to improve the light resistance of DAPI by using a discoloration inhibitor such as 1,4-diazabicyclo[2.2.2]octane (DABCO) or ProLong Gold (registered trademark, manufactured by Molecular Probes Inc.). However, although the use of such discoloration inhibitor alleviates the deterioration of the fluorescent label, since it cannot completely inhibit the deterioration, observation must be made in a short period.
Nevertheless, since the use of an oil-based mounting medium in staining with a fluorescent dye has the above-described problems, oil-based mounting media have not been used in staining with a fluorescent dye. Therefore, there is no motivation to admix a discoloration inhibitor to an oil-based mounting medium and to use the oil-based mounting medium in fluorescent staining, and such an attempt has thus not been made.